why cant get the band? - (Aug/25/2011 )
I badly need your guys help.
I am doing a reverse transcriptase PCR to amplify a gene from cDNA.
this gene expresses very well in the form of protein, which shows a very strong band by western blot.
however, after amplification by pcr, i barely detected the band. I use a plasmid of this gene as templet to amplify with the same primers, it shows very strong band as a positive control, indicating the system works, but why i cant get the band from cDNA amplification product, given the fact that it is a quite abundant expressed protein? can anyone help me to tackle this issue, i appreciate much.
Most likely your RNA is degraded or your RT reaction was not successful. Are you be able to amplify another gene from the cDNA template, for example, a housekeeping gene? Have you checked your RNA quality by running an agarose gel?
thanks Moderator, yeh the GAPDH looks perfect, :-(((( cant figure out why
please be aware that because housekeeping gene expression is very abundant, and can be amplified even there is RNA degradation. While other low expressing genes may not.
i see. i have just checked the concentration of the RNA product, it is still fine.
meanwhile i will check another gene beside housekeeping gene as well. how to check if rna is degraded? thanks